U.S. patent number 6,569,442 [Application Number 09/833,284] was granted by the patent office on 2003-05-27 for preparation of polymer foam having gelled sol coating for intervertebral disc reformation.
This patent grant is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to Paul Ducheyne, Jean Chin Chin Gan, Irving Shapiro, Edward Vresilovic.
United States Patent |
6,569,442 |
Gan , et al. |
May 27, 2003 |
Preparation of polymer foam having gelled sol coating for
intervertebral disc reformation
Abstract
Methods and materials are provided for reforming degenerated
intervertebral discs of the spine of a vertebrate and in particular
the spine of a human. Tissue is evacuated from the nucleus pulposus
portion of a degenerated intervertebral disc space, and a
biodegradable substrate containing isolated intervertebral disc
cells is implanted in the evacuated space for reformation of
intervertebral disc tissue. Intervertebral disc cells are provided
by digesting intervertebral disc tissue with collagenase and
incubating the cells in a medium supplemented with hyaluronidase.
The substrate may be bioactive so as to enhance cell function.
Substrates include bioactive glass granules, polymer foams and the
polymer foams coated with a bioactive gel material produced by a
sol-gel process. A polymer foam is dipped into a sol resulting from
combining a metal alkoxide precursor with water and acid, residual
sol is evacuated from pores of the foam, and remaining sol
contained by the foam is allowed to gel. The polymer of the foam
may be poly(D,L-lactide-co-glycolide). A preferred bioactive gel
material contains 70% SiO.sub.2, 25% CaO and 5% P.sub.2
O.sub.5.
Inventors: |
Gan; Jean Chin Chin (Ardmore,
PA), Ducheyne; Paul (Rosemont, PA), Vresilovic;
Edward (Philadelphia, PA), Shapiro; Irving
(Philadelphia, PA) |
Assignee: |
The Trustees of the University of
Pennsylvania (Philadelphia, PA)
|
Family
ID: |
24787795 |
Appl.
No.: |
09/833,284 |
Filed: |
April 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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314511 |
May 19, 1999 |
6240926 |
|
|
|
694191 |
Aug 8, 1996 |
5964807 |
|
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Current U.S.
Class: |
424/426; 128/898;
424/93.7; 435/180; 435/177; 435/176; 424/423; 435/325; 623/17.16;
435/397; 435/395; 435/381 |
Current CPC
Class: |
A61F
2/442 (20130101); A61L 27/56 (20130101); A61L
27/44 (20130101); A61L 27/3895 (20130101); A61L
27/025 (20130101); A61L 27/18 (20130101); A61L
27/306 (20130101); A61L 27/3817 (20130101); A61L
27/3856 (20130101); A61L 27/18 (20130101); A61L
27/306 (20130101); A61L 27/56 (20130101); A61L
27/18 (20130101); C08L 67/04 (20130101); A61F
2/30767 (20130101); A61L 2430/38 (20130101); A61F
2002/30062 (20130101); A61F 2002/30187 (20130101); A61F
2210/0004 (20130101); A61F 2230/0034 (20130101) |
Current International
Class: |
A61F
2/44 (20060101); A61L 27/02 (20060101); A61L
27/38 (20060101); A61L 27/44 (20060101); A61L
27/56 (20060101); A61L 27/18 (20060101); A61L
27/00 (20060101); A61L 27/30 (20060101); A61F
2/30 (20060101); A61F 2/02 (20060101); A61F
2/00 (20060101); A61F 002/44 (); A01N 063/02 ();
C12N 011/14 (); C12N 011/08 (); C12N 005/08 () |
Field of
Search: |
;435/174,176,177,180,325,378,381,395,397 ;424/93.7,423,426 ;128/898
;623/17.16 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Baldick, H. et al., "Bioactive Glass Increases Alkaline Phosphatase
Activity in Rat Marrow Stromal Cells In Vitro," Transactions 5th
World Biomaterials Conference, Toronto, Canada, May 29-Jun. 2,
1996. .
El-Ghannam et al., "Bioactive material template for in vitro
synthesis of bone," J. Biomed. Mat. Res., 1995, 29, 359-370. .
Healy et al., "Hydration and preferential molecular adsorption on
titanium in vitro," Biomaterials, 1992, 13(8), 553-561. .
Hedman et al., "Design of an Intervertebral Disc Prosthesis,"
Spine, 1991, 16(Supp. 6), 256-260. .
Hou et al., "Lumbar Intervertebral Disc Prosthesis," Chinese Med.
J., 1991, 104(5), 381-386. .
Lee et al., "Development of a Prosthetic Intervertebral Disc,"
Spine, 1991, 16(Supp. 6), 253-255. .
Lee et al., 35th Annual Meeting of the Orthopaedic Research
Society, Las Vegas, Nevada, Feb. 6-9, 1989. .
McMillin, C.R. et al., 20th Annual Meeting of the Society for
Biomaterials, Abstract, 1994. .
Qui, Q. et al., "Bone Growth on Sol-Gel Calcium Phosphate Thin
Films in Vitro," Cells and Materials, 1993, 3(4), 351-360. .
Sabolinski, "Cultured skin as a `smart material` for healing
wounds: experience in venous ulcers," Biomaterials, 1996, 17,
311-320. .
Schepers et al., "Bioactive glass particulate material as a filler
for bone lesions," J. Oral Rehab., 1991, 18, 439-452. .
Urbaniak et al., "Replacement of Intervertebral Discs in
Chimpanzees by Silicone-Dacron Implants: A Preliminary Report,"
Bio. J. Med. Mater. Res. Sym., 1973, 4, 165-168. .
White et al., Clinical Biomechanics of the Lumbar Spine, Churchill
Livingston, London, 1989. .
PCT International Search Report dated Oct. 29, 1997, 4
pages..
|
Primary Examiner: Naff; David M.
Attorney, Agent or Firm: Woodcock Washburn LLP
Parent Case Text
This Application is a division of application Ser. No. 09/314,511,
filed May 19, 1999, now U.S. Pat. No. 6,240,926, which is a
division of application Ser. No. 08/694,191, filed Aug. 8, 1996,
now U.S. Pat. No. 5,964,807.
Claims
What is claimed is:
1. A method of making a bioactive substrate comprising a porous
polymer foam coated with a bioactive gel material, wherein the
bioactive gel material is produced by a sol-gel process comprising
combining a metal alkoxide precursor with water and an acid
catalyst to produce a sol and allowing the sol to gel, comprising
the steps of: dipping the porous polymer foam into the sol whereby
pores of the polymer foam are filled with the sol, evacuating
residual sol from the pores of the polymer foam, and allowing
remaining sol contained by the porous polymer foam to gel to obtain
the porous polymer foam coated with the bioactive gel material,
wherein the bioactive gel material penetrates the pores of the
polymer foam and at least about 50% of the surface of the polymer
foam, which includes the pores, is coated with the bioactive gel
material.
2. The method of claim 1 wherein the pores of the polymer are
evacuated by creating a suction vacuum.
3. The method of claim 1 wherein the porous polymer foam is
polyglycolide (PGA), poly(D,L-lactide) (D,L-PLA) poly(L-lactide)
(L-PLA), poly(D,L-lactide-co-glycolide) (D,L-PLGA),
poly(L-lactide-co-glycolide) (PLGA), polycaprolatone (PCL),
polydioxanone, polyesteramides, copolyoxalates, polycarbonates, or
combinations thereof.
4. The method of claim 3 wherein the porous polymer foam is
poly(D,L-lactide-co-glycolide).
5. The method of claim 1 wherein the bioactive gel material is
comprised of from about 60% to about 100% SiO.sub.2, up to about
40% CaO; and up to about 10% P.sub.2 O.sub.5.
6. The method of claim 1 wherein the bioactive gel material has the
nominal composition 70% SiO.sub.2, 25% CaO; and 5% P.sub.2
O.sub.5.
7. The method of claim 1 wherein the bioactive gel material has a
pore size of leass than about 850 .mu.m.
8. The method of claim 1 wherein the bioactive gel material has a
percent density less than about 80%.
9. The method of claim 1, further comprising the step of adding
cell growth enhancers.
Description
FIELD OF THE INVENTION
The present invention concerns methods and materials useful for
reforming degenerated discs of the spine of a vertebrate and in
particular the spine of a human.
BACKGROUND OF THE INVENTION
Back pain is one of the most frequently reported musculoskeletal
problems in the United States. 80% of the adults will miss work at
least three times in their career due to back pain. The most common
factor causing low back pain is the degeneration of the disc. At
the ages between 35 to 37, approximately a third of the US
population have suffered from a herniated disc.
The main functions of the spine are to allow motion, transmit load
and protect the neural elements. The vertebrae of the spine
articulate with each other to allow motion in the frontal, sagittal
and transverse planes. As the weight of the upper body increases,
the vertebral bodies which are designed to sustain mainly
compressive loads, increase in size caudally. The intervertebral
disc is a major link between the adjacent vertebrae of the spine.
The intervertebral disc, the surrounding ligaments and muscles
provide stability to the spine.
The intervertebral discs make up about 20-33% of the lumbar spine
length. They are capable of sustaining weight and transferring the
load from one vertebral body to the next, as well as maintaining a
deformable space to accommodate normal spine movement. Each disc
consists of a gelatinous nucleus pulposus surrounded by a
laminated, fibrous annulus fibrosus, situated between the end
plates of the vertebrae above and below.
The nucleus pulposus contains collagen fibrils and water-binding
glycosaminoglycans. At birth, the nucleus pulposus contains 88%
water, however, this percentage decreases with age. This water loss
decreases its ability to withstand stress. The annulus fibrosus
consists of fibrocartilaginous tissue and fibrous protein. The
collagen fibers are arranged in between 10 to 20 lamellae which
form concentric rings around the nucleus pulposus. The collagen
fibers within each lamella are parallel to each other and runs at
an angle of approximately 60 degrees from vertical. The direction
of the inclination alternates with each lamellae. This crisscross
arrangement enables the annulus fibrosus to withstand torsional and
bending loads. The end-plates are composed of hyaline cartilage,
and are directly connected to the lamellae which form the inner
one-third of the annulus.
When under compressive loads, the nucleus pulposus flattens and
bulges out radially. The annulus fibrosus stretches, resisting the
stress. The end-plates of the vertebral body also resist the
ability of the nucleus pulposus to deform. Thus, pressure is
applied against the annulus and end-plate, transmitting the
compressive loads to the vertebral body. When tensile forces are
applied, the disc is raised to a certain height straining the
collagen fibers in the annulus. At bending, one side of the disc is
in tension while the other side is in compression. The annulus of
the compressed side bulges out.
When the disc is subjected to torsion, there are shear stresses
which vary proportionally to the distance from the axis of
rotation, in the horizontal and axial plane. The layer of fibers
oriented in the angle of motion is in tension while the fibers in
the preceding or succeeding layer are relaxed. Similarly in
sliding, the fibers oriented in the sliding direction are in
tension while the fibers in the other layers relax.
Repeated rotational loading initiates circumferential tears in the
annulus fibrosus, which gradually form radial tears into the
nucleus pulposus until the nucleus degrades within the disc. In
addition to the water loss which occurs with age, more water is
also lost due to nucleus rupture, thereby reducing its ability to
resist compressive loads. As such, the annulus bulges. As the
severity of the tear increases, much of the contents of the disc is
lost leaving a thin space of fibrous tissue. This condition is
called disc resorption.
Increasing disc collapse can cause facet subluxation and stenosis
of the intervertebral foramen. Subsequently, the degenerative
process involves the facet joints equally. As the annulus bulges
out posteriorly into the spinal canal, the nerve root may be
compressed causing sciatica. Pain is felt from the lower back to
the buttocks and the leg. Following the rupture of the disc,
excessive motions such as excessive extension or flexion can occur,
resulting in spine segmental instability. The spine is thus more
vulnerable to trauma. Herniation can occur due to disc degeneration
or excessive load factors especially compression. Pain may result
due to nerve root compression caused by protrusions.
The unstable phase of the degeneration progress allowing excessive
movement may result in degenerative spondylolisthesis, which is a
breakdown of posterior joints. The nerve is trapped between the
inferior articular facet of the vertebrae above and the body of
that below. Thus, sliding of a vertebral body on another damages
the posterior joints due to fatigue and apply traction on the nerve
root causing pain.
Surgical treatments for herniated disc include laminectomy, spinal
fusion and disc replacement with protheses.
At this time, 150,000 spinal fusion procedures are performed per
year in the U.S. alone, and the numbers are growing exponentially.
However, the results of spinal fusions are very varied. Some of the
effects include non-unions, slow rate of fusion even with
autografts, and significant frequency of morbidity at the graft
donor site. In addition, even if the fusion is successful, joint
motion is totally eliminated. Adverse effects of spinal fusions
have also been reported on adjacent unfused segments such as disc
degeneration, herniation, instability spondylolysis and facet joint
arthritis. A long-term follow-up of lower lumbar fusions in
patients from 21 to 52 years of age found that 44% of patients with
spinal fusions were currently still experiencing low-back pain and
57% had back pain within the previous year. 53% of the patients
tracked were on medication, 5% had late sequela secondary surgery,
15% had a repeat lumbar surgery, 42% had symptoms of spinal
stenosis, and 45% had instability proximal to their fusion. This
clinical data shows that significant long-term limitations are
associated with spinal fusion.
An alternative to spinal fusion is the use of an intervertebral
disc prosthesis. Ideally, a successful disc prosthesis will
simulate the function of a normal disc. The disc replacement must
be capable of sustaining weight and transferring load from one
vertebral body to the next. It should be robust enough not to be
injured during movement and should maintain a deformable space
between the vertebral body to accommodate movement.
Disc protheses should last for the lifetime of the patient, should
be able to be contained in the normal intervertebral disc space,
should have sufficient mechanical properties for normal body
function, should be able to be fixed to the vertebrae adjacent to
the disc, should be possible to implant, should not cause any
damage should the disc fail, and should be biocompatible.
There are at least 56 artificial disc designs which have been
patented or identified as being investigated, McMillin C. R. and
Steffee A. D., 20th Annual Meeting of the Society for Biomaterials
(abstract) (1994), although not all these devices have actually
been made or tested. They can be divided into two main categories.
Lee et al., Spine, Vol. 16, 253-255(1991). A first class includes
devices for nucleus pulposus replacements which includes metal ball
bearing, a silicone rubber nucleus, and a silicone fluid filled
plastic tube. Devices for total or subtotal replacement of the disc
have also been proposed such as a spring system, low-friction
sliding surfaces, a fluid filled chamber, elastic disc prosthesis
and elastic disc encased in a rigid column.
An example of total disc replacement is described by Urbaniak et
al., Bio. J. Med. Mater. Res. Sym., Vol. 4, 165-186 (1973) who
developed and tested, using chimpanzees, an intervertebral disc
device made of a central silicone layer sandwiched between two
layers of Dacron embedded in the silicone. The open-mesh Dacron was
chosen to allow tissue ingrowth for fixation to the adjacent
vertebrae. While spinal mobility was restored and the device
tolerated by the host, due to inexact fit of the device, bone
resorption and reactive bone formation were observable. Loose
fibrous tissue also indicated possible movement of the device.
Hou et al., Chinese Medical Journal, Vol. 104(5), 381-386 (1991),
developed a disc implant made of silicone rubber which restored
normal disc function. However, the presence of fibrous tissue
surrounding the implant indicated possible movement of the
device.
The SB Charite intervertebral disc endoprosthesis, White and
Panjabi, Clinical biomechanics of the lumbar spine, Churchill
Livingstone, London (1989), which has been tested clinically, is
fabricated from a biconvex polyethylene core sandwiched between two
concave-molded titanium end-plates. However, the endoprosthesis
shows insufficient mechanical performance and unlikely long-term
bone fixation to the device.
Two types of disc prostheses were developed and evaluated by Lee et
al., 35th Annual Meeting of the Orthopaedic Research Society, Las
Vegas, Nev., Feb. 6-9, 1989; Dacron fiber-reinforced polyurethane
elastomer (reinforcement located for the annulus section), and a
prosthesis made from thermoplastic polymer which is increasingly
rigid moving from the nucleus out to the end-plates. Yet another
design is made of cobalt-chromium-molybdenum (Co--Cr--Mo) alloy by
Hedman et al., Spine, Vol. 16, 256-60 (1991).
U.S. Pat. No. 4,911,718 (Lee et al.), U.S. Pat. No. 5,002,576
(Fuhrmann et al.), U.S. Pat. No. 4,911,718 (Lee et al.) and U.S.
Pat. No. 5,458,642 (Beer et al.) also teach permanent
intervertebral disc endoprostheses for total disc replacement.
All of foregoing intervertebral disc prostheses, however, merely
replace all or a part of the disc with synthetic materials which
must remain in place ad infinitum. These prostheses are generally
permanent implants which require observation of long term biologic
responses throughout the life of the prothesis. Furthermore, discs
that are not comprised of biocompatible material may be rejected by
the patient.
Procedures by which the tissues of the intervertebral disc are made
to reform or replace the degenerated tissue of the intervertebral
disc, would be highly desirable and a significant improvement over
the current state of the art which presently use such permanent
implants. Although efforts at tissue-engineering have been
reported, no one has, until now, accomplished reformation of
intervertebral disc tissue.
Repair of skin tissue has been achieved. For instance, skin
deficiencies which arise in severely burnt patients or in decubitus
wounds of diabetic patients have been so treated. Sabolinski,
Biomaterials, Vol. 17, 311-320 (1996). Cells are seeded onto
templates of either resorbable or non-resorbable material. Once
tissue begins to form the templates are dressed onto the site in
need of treatment. Tissue engineering of the skin, however, is
significantly different from tissue engineering of the
intervertebral disc because tissue compositions differ
significantly. In addition, the mechanical requirements of
engineered skin tissue are significantly different from those of
intervertebral disc tissue.
Some intervertebral disc prostheses provide for regrowth of the
intervertebral disc and concurrent resorption of the prothesis. For
example, U.S. Pat. Nos. 4,772,287 and 4,904,260 (Ray et al.) teach
prosthetic discs having an outer layer of strong, inert fibers
intermingled with bioresorbable materials which attract tissue
ingrowth. However, this prosthesis is purely a synthetic material
at the time of implantation and does not include any cells or
developing tissue. In addition, it provides only partial resorption
and the problems associated with permanent implants remain.
U.S. Pat. Nos. 5,108,438 and 5,258,043 (Stone) teach a porous
matrix of biocompatible and bioresorbable fibers which may be
interspersed with glycosaminoglycan molecules. The matrix serves as
a scaffold for regenerating disc tissue and replaces both the
annulus fibrosus and nucleus pulposus. However, replacement of this
much tissue is a relatively invasive procedure which requires
lengthy recovery time. Furthermore, these matrices do not use any
cells to stimulate tissue recovery nor is there any incipient
tissue formation in this device at the time of implantation.
Various materials have been seeded with cells in order to
facilitate cell function including proliferation and extracellular
matrix synthesis. For instance, El-Ghannam, et al., Journal of
Biomedical Materials Research, Vol. 29, 359-370 (1974), teaches in
vitro synthesis of bone-like tissue using bioactive glass
templates. Schepers, et al., J. Oral Rehab., Vol. 18, 439-452
(1991), analyzed the use of bioactive glass as fillers for bone
lesions. Also, porous polymeric matrices have been used. The
polymers include poly(lactic acid), poly(glycolic acid) and their
co-polymers. However, these polymers have not been taught to be
appropriate substrates for intervertebral disc cells which until
now have not been used to seed implants of any sort.
Ideally, intervertebral disc treatment would guide and possibly
stimulate the reformation of the tissue of affected intervertebral
disc, especially nucleus pulposus and annulus fibrosus tissue. It
could also biodegrade while allowing concurrent nucleus pulposus
and annulus fibrosus tissue ingrowth, thereby providing for disc
regeneration. Such an intervertebral disc material which is
biodegradable while still satisfying the mechanical requirements of
an intervertebral disc, has not been available until now.
OBJECTS OF THE INVENTION
An object of the present invention is to provide a method of
inducing and/or guiding intervertebral disc reformation using
biodegradable support substrates.
In yet another object of the present invention is provided
biodegradable substrates useful for intervertebral disc tissue
reformation.
Still another object of the invention is to provide material useful
for guiding and/or stimulating intervertebral disc tissue
reformation.
Another object of the invention is to provide methods of culturing
intervertebral disc cells.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of an exemplary hybrid material of the
present invention.
SUMMARY OF THE INVENTION
In accordance with methods of the present invention there are
provided methods for repairing damaged or degenerated
intervertebral discs. These methods comprise evacuating tissue from
the nucleus pulposus portion of a degenerated intervertebral disc
space, preparing hybrid material by combining isolated
intervertebral disc cells with a biodegradable substrate, and
implanting the hybrid material in the evacuated nucleus pulposus
space. In accordance with methods of the invention intervertebral
disc cell growth is guided and/or stimulated and intervertebral
disc tissue is reformed.
Methods of culturing intervertebral disc cells are also provided in
some aspects of the invention whereby intervertebral disc tissue is
digested with collagenase and incubated in medium supplemented with
hyaluronidase.
In still other aspects of the invention biodegradable substrates
are provided comprising polymer foam coated with bioactive
materials, which substrates are useful for intervertebral disc
tissue reformation.
In yet another aspect of the invention are provided hybrid
materials for reforming degenerate intervertebral disc tissue. The
hybrid materials can be made in the form of shaped bodies
comprising biodegradable substrate and intervertebral
disc-cells.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to methods of inducing
intervertebral disc repair by reformation of intervertebral disc
tissue. By implanting a hybrid material comprising intervertebral
disc cells and a biodegradable support substrate into the
intervertebral disc space, ingrowth of intervertebral disc cells is
induced. Thus, the present invention provides methods of inducing
self-regeneration of viable tissue and functional joints.
Methods of the present invention are useful to treat vertebrates
suffering from degenerated intervertebral disc conditions, and in
particular may be used to treat humans with such conditions.
A degenerated intervertebral disc has lost or damaged some or all
of its intervertebral disc tissue, primarily including its nucleus
pulposus tissue, due to any number of factors discussed herein,
including age and stress due to rotational loading. Degenerated
discs vary in severity from bulging discs to herniated or ruptured
discs. Patients suffering from a degenerated disc experience a
number of symptoms which include pain of the lower back, buttocks
and legs and may also include sciatica and degenerative
spondylolysis. In accordance with methods of the present invention
reformation or regeneration of intervertebral disc tissue occurs in
situ, replacing lost or damaged tissue and resulting in
amelioration or elimination of the conditions associated with the
degenerated disc.
In accordance with the present invention hybrid materials used to
induce and/or guide reformation of intervertebral disc tissue
comprise biodegradable substrates. Biodegradable means that the
substrate degrades into natural, biocompatible byproducts over time
until the substrate is substantially eliminated from the
implantation site and, ultimately, the body. Preferably in
accordance with methods of the present invention, the rate of
biodegradation of the substrate is less than or equal to the rate
of intervertebral disc tissue formation such that the rate of
tissue formation is sufficient to replace the support material
which has biodegraded.
In some aspects of the present invention the biodegradable
substrate may be bioactive. Bioactive, as used herein, is meant to
refer to substrates which enhance cell function as compared to cell
function of the same cell type in the absence of the substrate. For
instance, bioactive glass granules have been shown to enhance cell
growth of typical bone cells. Schepers et al., U.S. Pat. No.
5,204,106. In addition, dense bioactive glass discs have been found
to enhance osteoprogenitor cell differentiation beyond even those
levels of enhanced differentiation elicited by bone morphogenic
protein. H. Baldick, et al., Transactions 5th World Biomaterials
Conference, Toronto, II-114 (June, 1996).
The biodegradable substrate must also have sufficient mechanical
strength to act as a load bearing spacer until intervertebral disc
tissue is regenerated. In addition, the biodegradable substrate
must be biocompatible such that it does not elicit an autoimmune or
inflammatory response which might result in rejection of the
implanted hybrid material.
Biodegradable support substrates useful in methods of the present
invention include bioactive glass, polymer foam, and polymer foam
coated with sol gel bioactive material.
In accordance with some methods of the present invention bioactive
glass is employed as a substrate. Bioactive glass is described in
U.S. Pat. No. 5,204,104, incorporated by reference herein in its
entirety. The bioactive glass contains oxides of silicon, sodium,
calcium and phosphorous in the following percentages by weight:
about 40 to about 58% SiO.sub.2, about 10 to about 30% Na.sub.2 O,
about 10 to about 30% CaO, and 0 to about 10% P.sub.2 O.sub.5. In
preferred embodiments of the invention the nominal composition of
bioactive glass by weight is 45% SiO.sub.2, 24.5% Na.sub.2 O, 24.5%
CaO and 6% P.sub.2 O.sub.5 and is known as 45S5 bioactive glass.
Bioactive glass may be obtained from commercial sources such as
Orthovita, Inc. (Malvern, Pa.).
Granule size of the bioactive glass is selected based upon the
degree of vascularity of the affected tissue and generally will be
less than about 1000 .mu.m in diameter. In some embodiments of the
present invention it is preferred that the bioactive glass granules
be from about 200 .mu.m to about 300 .mu.m in diameter. In still
other embodiments of the present invention granule size is from
about 50 .mu.m to about 100 .mu.m.
In some embodiments of the present invention bioactive glass has
pores. Percent density (100%-percent porosity) of less than about
80% may be used in some aspects of the invention. Percent density
of about 10% to about 68% can be used for other aspects of the
invention. In some aspects of the present invention the pore size
should be less than about 850 .mu.m in diameter while about 150
.mu.m to about 600 .mu.m pore diameter is preferred.
One method of preparing porous bioactive glass is by mixing
bioactive glass granules of a desired size with sieved sacrificial
agent camphor particles of a desired amount and size. The camphor
sublimates during sintering leaving pores in the sintered glass.
Thus, the average particle size and weight percent of the camphor
particles is chosen to optimize the pore size and percent porosity,
respectively, of the glass. In some aspects of the invention
particle size may be less than about 850 .mu.m in diameter while
about 150 .mu.m to about 600 .mu.m is preferred. Thereafter the
glass may be treated with any aqueous buffer solution containing
ions, the identity and concentration of which is found in
interstitial fluid. Said treatments result in the formation of a
calcium phosphate rich layer at the glass surface. Typical buffers
include those prepared as described by Healy and Ducheyne,
Biomaterials, Vol. 13, 553-561 (1992), the subject matter of which
is incorporated herein by reference in its entirety.
In still other aspects of the present invention the support
substrate comprises polymer foam. Polymer foam useful in these
aspects of the invention are biocompatible and include
polyglycolide (PGA), poly(D,L-lactide) (D,L-PLA), poly(L-lactide)
(L-PLA), poly(D,L-lactide-co-glycolide),
(D,L-PLGA),poly(L-lactide-co-glycolide) (L-PLGA), polycaprolatone
(PCL),polydioxanone, polyesteramides, copolyoxalates, and
polycarbonates. D,L-PLGA, which is preferred in some embodiments of
the invention, may comprise 50% polylactide and 50% polyglycolide.
About 75% polylactide and about 25% polyglycolide is still more
preferred although it is anticipated that ratios may be varied to
optimize particular features of the individual polymers. For
instance, the mechanical strength of a polymer may be adjusted by
varying the percentage of PLA and the percentage of PGA may be
adjusted to optimize cell growth.
In some aspects of the invention polymer foam is coated with sol
gel bioactive material. Sol gel materials include glasses and
ceramics. Such bioactive compound are prepared by mixing a desired
polymer foam with NaCl to create the desired porosity and pore
size. Thereafter, the polymer, including the pores and interstices,
is coated with sol gel material.
Sol gel glass is prepared by combining a metal alkoxide precursor
with water and an acid catalyst to produce a gel. A typical process
is described in U.S. Ser. No. 08/477,585 (U.S. Pat. No. 5,591,453)
which is incorporated by reference herein in its entirety. Once
dried, the gel consists mostly of metal oxide with a glass
consistency. Sol gel bioactive material may be comprised of from
about 60 to about 100% silicon dioxide, up to about 40% calcium
oxide and up to about 10% diphosphorous pentoxide. A final product
of 70% SiO.sub.2, 25% CaO, and 5% P.sub.2 O.sub.5 is preferred in
some methods of the present invention although the concentration of
each may be adjusted to optimize critical features of the sol gel.
Other sol gel materials may be prepared by methods known in the
art. For instance, Qui, Q., et al., Cells and Materials, Vol. 3,
351-60 (1993), incorporated by reference herein in its entirety,
discusses methods of preparing calcium phosphate sol gel bioactive
material.
To coat the polymer, the polymer foam is dipped into the sol during
the sol gelation phase. The sol-filled foam is then placed in a
syringe filter and the sol is pulled through the foam by creating a
vacuum below using the syringe. Thus, the polymer is substantially
coated with sol gel, with residual sol gel being evacuated from the
polymer matrices. While it is preferred that most or all of the
polymer surfaces, including the surfaces of the pores and
interstices, be coated with sol gel bioactive glass, polymers which
are only partially coated with sol gel bioactive glass may also be
useful in some aspects of the present invention. It is desired in
some embodiments of the invention that greater than about 50% of
the polymer surface be coated.
To prepare the hybrid material, intervertebral disc cells are
combined with biodegradable substrate material. Intervertebral disc
cells may be isolated from tissue extracted from any accessible
intervertebral disc of the spine. For instance, tissue may be
extracted from the nucleus pulposus of lumbar discs, sacral discs
or cervical discs. Preferably, intervertebral disc cells are
primarily nucleus pulposus cells. In some embodiments it is
preferred that disc cells are at least 50% nucleus pulposus cells
while 90% nucleus pulposus cells is still more preferred. Cells may
be obtained from the patient being treated, or alternatively cells
may be extracted from donor tissue.
The present invention provides advantages over prior art methods in
that the entire degenerated disc need not be removed to treat a
degenerated disc. Rather, only the nucleus pulposus tissue need be
evacuated from the degenerated intervertebral disc. Degenerated
nucleus pulposus refers to a region of the intervertebral disc
where the tissue has severely reduced mechanical properties or
which has lost some or most of the nucleus pulposus tissue. The
present invention thus provides a less invasive procedure than that
of the prior art. In addition, the methods and hybrid materials of
the present invention prompt biological repair of normal tissue in
the disc which will result in better long term results than that
obtained with synthetic prostheses.
Evacuation of the degenerated intervertebral disc tissue, and
primarily the nucleus pulposus tissue, is performed using known
surgical tools with procedures developed to meet the needs of the
present invention. Generally an incision or bore is made at the
lateral edge in the annulus fibrosus and the intervertebral disc
tissue is extracted from the nucleus pulposus via, for example, the
guillotine cutting approach. The tissue may be extracted using a
scalpel, bore, or curette. Alternatively, tissue may be aspirated.
Ideally, the annulus fibrosus, or significant portions thereof, are
left intact. It is preferred for instance, that at least 50% of the
annulus fibrosus remain intact. It is still more preferred that at
least 85% of the annulus fibrosis remain intact. Arthroscopic
techniques are most preferred in accordance with methods of the
present invention.
Similar surgical techniques are utilized to extract intervertebral
disc tissue from other, non-degenerate intervertebral discs of the
spine of the patient or donor. For instance, similar techniques may
be used to obtain intervertebral tissue from sacral discs. Minor
modifications necessary to tailor the procedure to a particular
region of the spine would be appreciated by those skilled in the
art.
Where there is lag time between tissue evacuation and implantation
of the hybrid material, the evacuated space may be temporarily
filled with gel foam or other load bearing spacers known in the
art.
Intervertebral disc cells are isolated from extracted tissue.
Generally, tissue is fragmented and treated with enzymes such as
collagenase to disaggregate the cells into individual cells.
Preferably isolated cells are primarily nucleus pulposus cells with
50% nucleus pulposus cells being preferred and 90% nucleus pulposus
cells being more preferred. The cells are isolated using
centrifugation. Cells may then be combined with a biodegradable
substrate and implanted into the evacuated nucleus pulposus.
Alternatively, isolated intervertebral disc cells may be cultured
alone or seeded onto a biodegradable substrate and cultured
together with the biodegradable substrate for later
implantation.
In some aspects of the present invention the hybrid material may
also include factors to enhance cell growth. For instance,
TGF-.beta. and EGF may be added to the hybrid material to enhance
cell growth. Cells may be incubated alone or seeded on a substrate
in a tissue culture medium such as Dulbeccols Modified Eagle Medium
(DMEM) (pH 7.0), which may be supplemented with serum such as
heat-inactivate fetal bovine serum. Antifungal and antibacterial
agents may also be added. In preferred methods of the present
invention cells are incubated with about 0.5% to about 1.5%
hyaluronidase.
In some aspects of the present invention the end plate may be
partially decorticated to enhance vascularization. Thereafter,
cells may be implanted or alternatively, after cell attachment,
hyaluronidase is removed and incubation is resumed with a medium
supplemented with 0.001% ascorbic acid in the absence of
hyaluronidase. Medium supplemented with 0.0025% ascorbic acid is
used to replenish the cell solutions.
Hybrids of intervertebral disc cells and biodegradable substrate
may then be implanted into the evacuated intervertebral disc space
using surgical procedure such as described above.
Hybrid materials are provided by the present invention. Such
hybrids can then be shaped for insertion into the intervertebral
disc space of a patient. Exemplary FIG. 1 shows a shaped hybrid
material comprising biodegradable substrate and intervertebral
cells. Intervertebral cells are located on the outer surface 10 and
on the surfaces of the pores and interstices 12 of the shaped
substrate. It should be noted that FIG. 1 depicts one possible
embodiment and should not be construed as limiting the invention in
any way.
The substrate should generally have a rectangular shape. A
cylindrical pad shape is also envisioned.
The following examples are illustrative but are not meant to be
limiting of the present invention.
EXAMPLES
Example 1
Evacuation of Nucleus Pulposus
Mature New Zealand rabbits weighing 4-5 kg are used. For each
rabbit, L4-L5 or, when possible L4-L5 and L5-L6 disc spaces are
accessed as those are the biggest sections. The anesthetics
Ketamine, HCl 30 mg/kg, and Xylazine 6 mg/kg, are administered
intramuscularly. Using a paraspinal posterolateral splitting
approach, the large cephalad-facing transverse process of the
lumbar spine is identified and removed with a rongeur. The
intervertebral disc can then be seen. An incision is made in the
annulus fibrosus. Using a high-power surgical microscope, the
nucleus pulposus tissue is scraped out carefully with a curette.
The space is then packed with gel foam. The rabbit is closed
provisionally.
Example 2
Isolation of Intervertebral Disc Cells
Intervertebral disc tissue is obtained as described in Example 1 or
from an amputated tail section. Under aseptic condition, the
intervertebral disc tissue is diced with a scalpel and placed in a
T25 tissue culture flask with Dulbecco's Modified Eagle Medium
(DMEM) adjusted to pH 7.0, supplemented with 10% heat inactivated
fetal bovine serum and 1% penicillin/streptomycin (TCM). The tissue
is then treated with 0.25% collagenase for two hours at 37.degree.
C. An equal amount of TCM to collagenase is added to stop
treatment. The mixture is centrifuged at 1000 r/min for 10 minutes
and supernatant is discarded. TCM is added and the mixture is
filtered to remove debris. The mixture is again centrifuged and
supernatant discarded. Cells are resuspended in TCM supplemented
with 1% hyaluronidase (400 u/ml).
Example 3
Culture of Intervertebral Disc Cells
Cells are cultured in TCM supplemented with 1% hyaluronidase (400
u/ml) at 37.degree. C. in 5% CO2/95% air. Once cells attach medium
is changed to TCM supplemented with 0.001% ascorbic acid in the
absence of hyaluronidase. Cells are resuspended in fresh medium
supplemented with 0.0025% ascorbic acid every 3 days.
Example 4
Preparation of Bioactive Glass
Bioactive glass granules (45S5) having diameters of 40 .mu.m to 71
.mu.m can be obtained from Orthovita, Inc. (Malvern, Pa.). Prior to
implantation or addition to cell culture, the specimens are
sterilized in ethylene oxide.
Example 5
Preparation of Sintered Porous Bioactive Glass
Bioactive glass granules having diameters of 40 .mu.m to 71 .mu.m
can be obtained from Orthovita, Inc. (Malvern, Pa.). The glass
granules are mixed with 20.2 weight % sieved sacrificial agent
camphor C.sub.10 H.sub.16 O with grain size of 300 .mu.m to 500
.mu.m. The mixture is mechanically mixed overnight, and cold
pressed at 350 MPa. The disc obtained is heat treated at
575.degree. C. for 45 minutes. The heating rate is 10.degree.
C./min. It is then left to cool at room temperature. The disc is
immersed in acetone for 30 minutes and dried at 37.degree. C. The
disc is cut to the desired dimensions using a diamond-wheel saw.
The disc is washed in acetone for 15 minutes. The specimen is then
conditioned in tris buffer with electrolytes added (TE)
(El-Ghannam, et al., Journal of Biomedical Materials Research, Vol.
29, 359-370 (1974)), for 2 days to obtain the desired formation of
calcium phosphate-rich layer at the glass surface. The specimen is
rinsed with methanol and dried at 37.degree. C. The specimen is
analyzed using scanning electron microscopy (SEM), Fourier
Transform Infrared (FTIR) spectroscopy and X-ray diffraction (XRD).
Prior to implantation or introduction to cell culture, the specimen
is sterilized in ethylene oxide.
Example 6
Preparation of Polymer Foam
3 g of NaCl with particle sizes 300 .mu.m to 500 .mu.m, and 2 g of
D,L-PLGA 75/25 (75% polylactide/25% polyglycolide) polymer foam
were mixed. The dispersion is vortexed and cast in a 5 cm petri
dish. The solvent is allowed to evaporate from the covered petri
dish for 48 hrs. To remove residual amounts of chloroform, the
petri dish is vacuum-dried at 13 Pa for 24 hrs. The material is
then immersed in 250 ml distilled deionized water at 37.degree. C.
for 96 hrs. The water is changed every 12 hrs to leach out the
salt. The salt-free membrane is air-dried for 24 hrs, followed by
vacuum-drying at 13 Pa for 48 hrs. The material is then cut to the
desired geometry with a razor blade. The membrane is stored in a
desiccator under vacuum. The specimens are analyzed using SEM. The
specimen will have 60% pore density with pore sizes 300 to 500
.mu.m. Prior to implantation or introduction into cell culture, the
specimens are sterilized in ethylene oxide.
Example 7
Preparation of Polymer Foam coated with Sol Gel Bioactive Glass
Tetramethylorthosilane (TMOS),calcium methoxyethoxide and triethyl
phosphate are mixed for 5 minutes in an argon atmosphere using a
magnetic stirrer. Respective amounts of each are chosen such that
the resulting product is 70% SiO.sub.2 -25%CaO-5%P.sub.2 O.sub.2
(upon drying). They are mixed using a magnetic stirrer for 5 min.
The PLGA polymer foam prepared according to Example 5 is dipped
into the sol approximately halfway to gelation. The foam is dipped
2 to 3 times to make sure that the sol completely fills the polymer
foam. The sol-filled foam is then placed in a syringe filter with
appropriate filter pore size which only allows the sol to flow
through. This syringe filter is attached to a syringe. The sol is
pulled through the foam by creating a vacuum below using the
syringe. EDAX and SEM are used to analyze pore size, porosity and
the thickness/uniformity of the sol gel bioactive glass coating.
Prior to implantation or introduction to cell culture, the
specimens are sterilized in ethylene oxide.
Example 8
Cell Phenotype
Cell phenotype of cells cultured in accordance with the method of
Example 3 is examined. Immunofluorescent staining of cells shows
positive staining for proteoglycan and collagen type II, markers of
intervertebral disc cell phenotype. Substantially negative staining
for collagen I, a annulus fibrosus marker, was also observed.
Example 9
Cell Reversion
Intervertebral disc cells cultured as described in Example 3 are
tested for reversion. Cells are placed in Eppendorf tube with TCM
and spun down to form a pellet. Cell histology is examined after 4,
8 and 12 days by washing the pellet and fixing it with 70% ethanol.
The cells are dehydrated, embedded and cut. The sample is stained
with hematoxylin-eosin and toluidine blue.
The histology of cultured cells is compared to the histology of
nucleus pulposus tissue prepared immediately upon retrieval.
Histology of the cells evidences a reversion to the original
morphology of the cells.
Example 10
Implantation of Biodegradable Substrate
Cells are prepared in accordance with Examples 2 and 3. Cells are
counted. Biodegradable substrates prepared as described in Examples
4-7 each placed in a tissue culture dish and immersed in TCM for 1
hour. The cells are seeded onto each of the sterile biodegradable
substrates prepared as described in Examples 4-7 in TCM with
hyaluronidase and left to attach for at least one hour before
flooding the dish with TCM. Cells are incubated overnight.
Attachment is detected using SEM.
The rabbit treated as described in Example 1 is reopened per
surgical technique described in Example 1, and the intervertebral
disc space accessed. The gel foam is retrieved and the
cell-biodegradable substrate hybrid material inserted in place. The
wound is closed.
Example 11
Effect on Neurological Function
Regular post-operative neurological functions are evaluated to
examine the subject for any spinal injury such as lameness. The
effect of the hybrid material on the behavior of the disc can be
observed and generally compared by taking radiographs of the spine
immediately pre-operation, post-operation and at 1 month time
periods until the animal is sacrificed.
Example 12
Histological Analysis
Histological analysis is performed to determine cell ingrowth, cell
types, tissue morphology, and absence of inflammation. To this end,
the retrieved disc is fixed in 70% ethanol and dehydrated. After
embedding in methyl methacrylate, sections are cut with a diamond
saw, ground, polished with silicon carbide paper and diamond paste,
and stained. Histology is done on normal discs and discs retrieved
at the various time periods. Analysis will show ingrowth of cells
with concurrent degradation of implanted hybrid material with
little to no inflammation.
* * * * *